CN112300957B - Bacillus halophilus and method for industrially producing ectoin by using same - Google Patents
Bacillus halophilus and method for industrially producing ectoin by using same Download PDFInfo
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- CN112300957B CN112300957B CN201911272352.1A CN201911272352A CN112300957B CN 112300957 B CN112300957 B CN 112300957B CN 201911272352 A CN201911272352 A CN 201911272352A CN 112300957 B CN112300957 B CN 112300957B
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Abstract
The invention discloses bacillus halophilus and a method for industrially producing ectoin by using the same. The invention firstly separates a strain with the secretion characteristic of ectoin from sunshine coastal sea mud, and then obtains bacillus halophilus (Halobacillus sp.) FL-2423 with high ectoin yield and stable genetic characteristic by mutation screening, wherein the preservation number of the strain is CGMCC No. 18783. The strain is moderately halophilic bacteria, and is subjected to fermentation culture in a culture medium with the NaCl concentration of 50-100g/L, ectoin is synthesized intracellularly, and the fermentation yield reaches 10-16 g/L.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus halophilus and a method for industrially producing ectoin by using the same.
Background
Ectoin, the chemical name of which is 1,4,5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid, also known as tetrahydropyrimidine, is a cyclic amino acid derivative, which is an osmotic pressure compensating solute synthesized by some microorganisms in response to environmental osmotic stress. The ectoin has wide application, not only has the functions of moisturizing, locking water and resisting wrinkles when being used in cosmetics, but also has the function of stabilizing the activity of cells and macromolecular substances, such as enzyme, nucleic acid stabilizer and the like. Meanwhile, the ectoin has wide application prospect and development value in the aspect of medicine, can be used as a protective agent for healthy cells in the chemotherapy process, and has certain prevention effect on senile dementia and Parkinson's disease.
Based on special physicochemical characteristics, strains with high ectoine yield are mostly screened from extreme environments such as high salt, reported high-yield strains are abundant in halophilous monads, marine coccus and bacillus, wild bacteria reported at home and abroad are used for preparing ectoine, the yield is low, the later extraction process is complex, required equipment and operation steps are strict, and the cost is high, so that the cost of the ectoine sold in the market at present is high, the use range of the ectoine is limited, and consumers have to find substitutes with good effect and low cost for the ectoine.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a bacillus halophilus (Halobacillus sp.) FL-2423, which is a moderately halophilic bacterium, reduces the use concentration of salt in fermentation culture, is secreted in cells under the condition of moderately saline, and is beneficial to later extraction. In the fermentation process, sodium glutamate is adopted as a culture medium of a unique carbon source, and fermentation is carried out under the condition of lower salt concentration, so that the fermentation yield is high, and the requirement on the salt tolerance of fermentation equipment is low.
The invention firstly separates a strain with high growth speed, wide adaptive salt concentration range and the secretion characteristic of ectoine from sunshine coastal sea mud, and then carries out chemical and physical compound mutation breeding and screening on the strain to obtain a mutant strain, namely bacillus halophilus (Halobacillus sp.) FL-2423 with high ectoine yield and stable hereditary characteristic. The strain is preserved in China general microbiological culture Collection center (address: China academy of sciences, No. 3, West Lu No.1, Beijing, Chaoyang, North Cheng) on 11.01.2019, and the preservation number is CGMCC No. 18783. The bacterial colony characteristics of the strain are shown in figure 2, and the 16S rDNA sequence is shown in a sequence table SEQ No. 1.
The invention also provides a method for industrially producing the ectoin by adopting the bacillus halophilus (Halobacillus sp.) FL-2423, which is characterized in that,
1) seed liquid culture
Inoculating Bacillus halophilus (Halobacillus sp.) FL-2423 into a seed culture medium for shake cultivation at the culture temperature of 26-32 ℃ for 16-30 hours;
wherein, the components and contents of the seed culture medium are as follows: 20-50g/L of monosodium glutamate, 5-15g/L of dipotassium phosphate, 1-5g/L of monopotassium phosphate, 0.2-1g/L of magnesium sulfate, 0-0.05g/L of manganese sulfate, 1-10g/L of yeast powder, 20-60g/L of sodium chloride and pH 7.0.
Preferably, the seed culture medium comprises the following components in percentage by weight: 25g/L of monosodium glutamate, 5g/L of dipotassium phosphate, 3g/L of monopotassium phosphate, 0.4g/L of magnesium sulfate, 0.01g/L of manganese sulfate, 1g/L of yeast powder, 30g/L of sodium chloride and pH 7.0.
2) Aerobic fermentation
Inoculating the seed solution into a fermentation culture medium at an inoculation amount (volume ratio) of 5-10%, and culturing at 25-30 deg.C for 24-96 hr to obtain intracellular fermentation broth for producing ectoin.
Wherein the components of the fermentation medium comprise a carbon source, inorganic salt and a nitrogen source, wherein sodium glutamate is used as the only carbon source. The inorganic salt comprises dipotassium hydrogen phosphate, potassium dihydrogen phosphate and sodium chloride, and also can comprise magnesium sulfate, manganese sulfate and ammonium sulfate, and a small amount of nitrogen source is yeast powder or peptone. Wherein the concentration of sodium chloride suitable for syncytium endocrine ectoine is: 50-100 g/L.
Preferably, the fermentation medium comprises the following components in percentage by weight: 10-50g/L of sodium glutamate, 5-10g/L of dipotassium phosphate, 1-5g/L of monopotassium phosphate, 0-1g/L of magnesium sulfate, 0-0.1g/L of manganese sulfate, 50-100g/L of sodium chloride, 0-20g/L of ammonium sulfate, 5-20g/L of yeast powder or peptone and pH 7.0.
Further preferably, the fermentation medium comprises the following components in percentage by weight: 40g/L of sodium glutamate, 8g/L of dipotassium phosphate, 4g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 10g/L of yeast powder, 60g/L of sodium chloride, 10g/L of ammonium sulfate and pH 7.0.
The invention has the beneficial effects that:
1. the bacillus halophilus (Halobacillus sp.) FL-2423 is moderately halophilic bacteria, reduces the salt use concentration in fermentation culture, finds the balance of intracellular ectoine salt, secretes the intracellular ectoine salt under the moderately saline condition, and is beneficial to later extraction (most of the ectoine is secreted outside the cell under the low-salt condition and is not beneficial to extraction).
2. In the fermentation process, sodium glutamate is used as a culture medium of a unique carbon source, and fermentation is carried out under the condition of low salt concentration, so that the fermentation yield reaches 10-16g/L, and compared with the wild bacteria reported at present, the yield of the ectoin is in the leading position.
3. The invention reduces the salt concentration in the culture medium and the corrosion of high salt to fermentation equipment and the pollution to the environment.
Drawings
FIG. 1 is a graph showing the growth of a mutant strain obtained according to the present invention;
FIG. 2 is a characteristic diagram of a colony of Bacillus halophilus (Halobacillus sp.) FL-2423; wherein the right image is an enlarged view of the left image;
FIG. 3 is a graph showing the change in the yield of ectoin in 100L fermentation culture with time.
Detailed Description
Example 1: obtaining of Strain
Diluting a sea mud sample collected in a sunshine coastal sea area in sterile water, coating a diluent on culture medium plates containing different NaCl concentrations, culturing at 28 ℃, selecting a single bacterial colony after the bacterial colony grows out, continuously separating and purifying until obtaining pure bacteria, primarily screening bacterial strains with high growth speed and wide applicable salt concentration range, secondarily screening bacterial strains with High Performance Liquid Chromatography (HPLC) method for measuring the yield of the ectoine, and screening the bacterial strains with the yield of the ectoine of 2.4g/L in a laboratory.
The strain is subjected to chemical and physical compound mutation breeding, and is screened in a large amount in a laboratory to obtain the mutant strain FL-2423 with high yield of ectoin, the mutant strain has the capability of industrially producing ectoin and stable genetic characteristics, the growth curve of the mutant strain is shown in figure 1, the shake flask fermentation yield is 12g/L, and the yield is improved by 5 times compared with that of the original strain.
Example 2: amplification, sequencing and sequence comparison of 16SrDNA of strain
Collecting fresh thallus, extracting total DNA template with Ezup column type bacterial genome DNA extraction kit, amplifying 16S rDNA gene with universal primer, detecting and purifying PCR product, and direct sequence determination with Shanghai biological engineering technology company.
The primer sequences are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively;
1495R：5’-GGTTACCTTGTTACGACTT-3’；
the 10 μ L PCR reaction was as follows: 1 mu L of template; 1 mu L of each of the upstream primer and the downstream primer; dream Taq Green PCR Master Mix 5. mu.L; ddH2O 2μL。
PCR conditions were as follows: 5min at 95 ℃, 30s at 94 ℃, 2min at 52 ℃ and 1min at 72 ℃; the cycle was 30 times.
The full sequence results were determined as follows: the 16S rDNA sequence of the strain is shown in a sequence table SEQ No.1, and the colony characteristics of the mutant strain are shown in figure 2. And uploading the obtained full sequence to an NCBI website for sequence comparison, and finally determining the variant strain FL-2423 as Bacillus halobacter sp. The strain is preserved in China general microbiological culture Collection center (address: China academy of sciences, No. 3, West Lu No.1, Beijing, Chaoyang, North Cheng) on 11.01.2019, and the preservation number is CGMCC No. 18783.
Example 3: method for culturing bacillus halophilus (Halobacillus sp.) FL-2423
1) Inoculating Bacillus halophilus (Halobacillus sp.) FL-2423 into a seed culture medium, and culturing at 28 deg.C for 24h to obtain seed solution.
Seed culture medium components: 25g/L of monosodium glutamate, 5g/L of dipotassium phosphate, 3g/L of monopotassium phosphate, 0.4g/L of magnesium sulfate, 0.01g/L of manganese sulfate, 1g/L of yeast powder, 30g/L of sodium chloride and pH 7.0.
2) Inoculating the seed solution into a shake flask with the inoculation amount of 5%, and culturing at 28 deg.C for 72h to obtain the product with the yield of ectoin in fermentation liquid of 12.5 g/L.
Fermentation medium components: 40g/L of sodium glutamate, 8g/L of dipotassium phosphate, 4g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 10g/L of yeast powder, 60g/L of sodium chloride, 10g/L of ammonium sulfate and pH 7.0.
Example 4:
the seed liquid cultured in example 3 was inoculated at an inoculation amount of 5% into a 100L fermenter containing 60L of a fermentation medium, at a tank pressure of not more than 0.1MPa and an aeration ratio of 15: after culturing for 72h at the temperature of 1 and 28 ℃, the yield of the ectoin in the fermentation liquor is 14 g/L.
The change in the yield of ectoin over time is shown in FIG. 3, and can be seen from FIG. 3: the yield is gradually increased along with the increase of time, and the yield reaches 14g/L at 72 h.
Example 5:
inoculating the seed solution cultured in example 3 in a 1T fermenter at 10% inoculum size, pH 7.0, culturing at 28 deg.C for 72 hr, at a tank pressure of 0.05-0.06 MPa, an initial rotation speed of 80rpm, and an aeration rate of 200m3And h, gradually adjusting the stirring speed and the ventilation quantity along with the fermentation process, adjusting the stirring speed and the ventilation quantity to the maximum within about 12h to keep the dissolved oxygen at more than 30 percent, and finally, the yield of the ectoin in the fermentation liquid is 14.5 g/L.
Example 6:
inoculating the seed solution cultured in example 3 in a 5T fermenter at 10% inoculation amount, pH 7.0, culturing at 28 deg.C for 72 hr, at a tank pressure of 0.05-0.06 MPa, at an initial rotation speed of 80rpm, and with an aeration amount of 200m3And/h, gradually adjusting the stirring speed and the ventilation quantity along with the fermentation process, adjusting the stirring speed and the ventilation quantity to the maximum within about 12h, keeping the dissolved oxygen at more than 30 percent, and controlling the yield of the ectoine in the fermentation liquid to be 16 g/L.
SEQUENCE LISTING
<110> Shandong Furuida Biotech Co., Ltd
<120> Bacillus halophilus and method for industrially producing ectoin by using same
<130> 0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1536
<212> DNA
<213> 16S rDNA sequence of Bacillus halophilus (Halobacillus sp.) FL-2423
<400> 1
cgtgactgag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc gcgggaagcg 60
agtggctccc ttcggggtga agctcgtgga acgagcggcg gacgggtgag taacacgtgg 120
gcaacctgcc tgtaagatcg gaataacccc gggaaaccgg ggctaatgcc gggtaatact 180
ttctttcgca tgaaggaaag ttgaaagatg gcttctagct atcacttaca gatgggcccg 240
cggcgcatta gctagttggt gaggtaacgg ctcaccaagg cgacgatgcg tagccgacct 300
gagagggtga tcggccacac tgggactgag acacggccca gactcctacg ggaggcagca 360
gtagggaatc ttccgcaatg gacgaaagtc tgacggagca acgccgcgtg aacgatgaag 420
gtcttcggat cgtaaagttc tgttgttagg gaagaacaag taccgtgcga atagagcggt 480
accttgacgg tacctaacga ggaagccccg gctaactacg tgccagcagc cgcggtaata 540
cgtagggggc aagcgttgtc cggaattatt gggcgtaaag cgcgcgcagg cggttcctta 600
agtctgatgt gaaagcccac ggctcaaccg tggagggtca ttggaaactg gggaacttga 660
ggacagaaga ggagagtgga attccacgtg tagcggtgaa atgcgtagat atgtggagga 720
acaccagtgg cgaaggcgac tctctggtct gtttctgacg ctgaggtgcg aaagcgtggg 780
tagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaggtgtta 840
gggggcttcc accccttagt gctgaagtta acgcattaag cactccgcct ggggagtacg 900
gccgcaaggc tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 960
tttaattcga agcaacgcga agaaccttac caggtcttga catccttgga catccctaga 1020
gatagggctt tcccttcggg gaccaagtga caggtggtgc atggttgtcg tcagctcgtg 1080
tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ctaatcttag ttgccagcat 1140
tcagttgggc actctaaggt gactgccggt gacaaaccgg aggaaggcgg ggatgacgtc 1200
aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggatg gtacaaaggg 1260
cagcgaagcc gcgaggtgta gcaaatccca taaaaccatt ctcagttcgg attgcaggct 1320
gcaactcgcc tgcatgaagc cggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa 1380
tacgttcccg ggccttgtac acaccgcccg tcacaccacg agagttggca acacccgaag 1440
tcggtgaggt aacctttttg gagccagccg ccgaaggtgg ggccaatgat tggggtgaag 1500
tcgtaacaag gtagccgtat cggaaggtgc gactcg 1536
Claims (9)
1. A strain of Bacillus halophilus (Halobacillus sp.) FL-2423 has a preservation number of CGMCC No. 18783.
2. Use of bacillus halodurans (Halobacillus sp.) FL-2423 according to claim 1 for the production of ectoin.
3. A method for industrially producing ectoin, characterized in that Bacillus halodurans (Halobacillus sp.) FL-2423 according to claim 1 is used for fermentation.
4. The method for industrially producing ectoin according to claim 3, wherein the fermentation medium used in the fermentation is a fermentation medium containing sodium glutamate as a sole carbon source; the inorganic salt comprises dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and sodium chloride; the sodium chloride concentration in the fermentation medium was: 50-100 g/L.
5. The method of claim 4, wherein the inorganic salt further comprises magnesium sulfate, manganese sulfate, ammonium sulfate.
6. The method for the industrial production of ectoin according to claim 4, wherein the fermentation medium comprises the following components by weight: 10-50g/L of sodium glutamate, 5-10g/L of dipotassium phosphate, 1-5g/L of monopotassium phosphate, 0-1g/L of magnesium sulfate, 0-0.1g/L of manganese sulfate, 50-100g/L of sodium chloride, 0-20g/L of ammonium sulfate, 5-20g/L of yeast powder or peptone and pH 7.0.
7. The method according to claim 6, wherein the step of producing ectoin is carried out,
1) seed liquid culture
Inoculating the Bacillus halophilus (Halobacillus sp.) FL-2423 strain of claim 1 into a seed culture medium for shake cultivation at 26-32 ℃ for 16-30 hours;
wherein, the components and contents of the seed culture medium are as follows: 20-50g/L of monosodium glutamate, 5-15g/L of dipotassium phosphate, 1-5g/L of monopotassium phosphate, 0.2-1g/L of magnesium sulfate, 0-0.05g/L of manganese sulfate, 1-10g/L of yeast powder, 20-60g/L of sodium chloride and pH 7.0;
2) aerobic fermentation
Inoculating the seed solution into a fermentation culture medium in an inoculation amount of 5-10% by volume, and culturing at 25-30 deg.C for 24-96 hr to obtain intracellular fermentation broth for producing ectoin.
8. The method according to claim 7, wherein the step of industrially producing ectoin,
the seed culture medium comprises the following components in percentage by weight: 25g/L of monosodium glutamate, 5g/L of dipotassium phosphate, 3g/L of monopotassium phosphate, 0.4g/L of magnesium sulfate, 0.01g/L of manganese sulfate, 1g/L of yeast powder, 30g/L of sodium chloride and pH 7.0.
9. The method according to claim 6 or 7, wherein the step of producing ectoin is carried out,
the fermentation medium comprises the following components in percentage by weight: 40g/L of sodium glutamate, 8g/L of dipotassium phosphate, 4g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 10g/L of yeast powder, 60g/L of sodium chloride, 10g/L of ammonium sulfate and pH 7.0.
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| CN115804381B (en) * | 2022-10-19 | 2023-06-16 | 山东福瑞达生物科技有限公司 | Application of tetrahydropyrimidine in relieving phytotoxicity of sulfonylurea herbicide to corn |
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| CN102517306B (en) * | 2011-12-16 | 2013-04-10 | 山东潍坊润丰化工有限公司 | Ectoine synthetase gene, recombinant vector, recombinant engineering bacterium and application of recombinant engineering bacterium |
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